The effectiveness of newly synthesized quaternary ammonium salts differing in chain length and type of counterion against priority human pathogens

Quaternary ammonium salts (QAS) commonly occur as active substances in disinfectants. QAS have the important property of coating abiotic surfaces, which prevents adhesion of microorganisms, thus inhibiting biofilm formation. In this study, a group of nine monomeric QAS, differing in the structure and length of the aliphatic chain (C12, C14, C16) and the counterion (methylcarbonate, acetate, bromide), were investigated. The study included an analysis of their action against planktonic forms as well as bacterial biofilms. The compounds were tested for their anti-adhesion properties on stainless steel, polystyrene, silicone and glass surfaces. Moreover, mutagenicity analysis and evaluation of hemolytic properties were performed. It was found that compounds with 16-carbon hydrophobic chains were the most promising against both planktonic forms and biofilms. Tested surfactants (C12, C14, C16) showed anti-adhesion activity but it was dependent on the type of the surface and strain used. The tested compounds at MIC concentrations did not cause hemolysis of sheep blood cells. The type of counterion was not as significant for the activity of the compound as the length of the hydrophobic aliphatic chain.


Results
Design, synthesis, identification, and characteristics of novel QAS surfactants. The design of novel alkylamide-type QAS surfactants took into account three main aspects: moderate biodegradability assuming both safety and sufficient stability during storage/usage; appropriate aqueous solubility, especially for the most effective long alkyl chain derivatives, as well as different counterion types, including "green" and reactive ones. Therefore, our rational products comprised an antimicrobial QAS headgroup, a tertiary amide linker without N-H bonds for moderation of hydrogen bonding, superior aqueous solubility, and balanced chemical stability/biodegradability as well as three different counterions: bromide, acetate and methylcarbonate. The design and some basic properties of the studied surfactants are shown in Fig. 1.
According to cationic surfactants described in the literature, we designed and synthesized novel antimicrobial cationic surfactants of linear (single-head, single-tail) structure with an appropriate cleavable tertiary amide linker as well as counterions meeting the requirements of safe disinfecting agents and the principles of "green chemistry". Our surfactants meet all above-mentioned aspects of novel multifunctional amphiphiles: synthetic requirements of sustainable chemistry with potential for technological applications, avoidance of toxic intermediates and excellent water solubility. Taking into consideration possible quaternizing agents, e.g. alkyl halide, dimethyl sulfate or dimethyl carbonate, we chose methyl bromide-a standard intermediate for QAS synthesis, enabling very mild synthetic conditions-and a very potent "green chemistry alternative": dimethyl carbonate. Moreover, monomethyl carbonates-yielded in reaction of appropriate hydrophobic intermediate with dimethyl carbonate-may constitute raw materials for surfactants with novel, biocompatible counterions, as acetate and lactate groups 16,17 . Generally, an excess of quaternizing agent is needed to achieve a sufficient reaction rate and appropriate yield of the desired product. That is why one of the main advantages of dimethyl carbonate as a quaternizing agent is its optimal boiling point (around 90 °C) enabling easy removal after reaction by evaporation Scientific Reports | (2022) 12:21799 | https://doi.org/10.1038/s41598-022-24760-y www.nature.com/scientificreports/ under reduced pressure, followed by reuse for synthesis as well as reduced risk of accidental evaporation due to reaction heat 27 . In contrast to dimethyl sulfate, a toxic, mutagenic, and carcinogenic quaternizing agent with moderate stability in the environment, dimethyl carbonate does not constitute a risk connected with its presence as an impurity in the given product. The structures of the obtained QAS-type surfactants were confirmed by 1 H NMR measurements, while values of critical micelle concentrations were assessed by conductrometric measurements (see detailed description in Electronic Supplementary Material, Figs. S10-S12). In general, all 1 H NMR spectra of the studied surfactants (see spectra in Electronic Supplementary Material, Figs. S13-S15 and Tables 3, 4, 5 for detailed information) showed characteristic signals, attributed to methyl groups at the end of the alkyl chain and within the tertiary amide linker (around 0.85-0.9 ppm and 3.05-3.1 ppm, respectively). Moreover, a strong singlet of three methyl moieties within the tertiary ammonium headgroup was visible at 3.3-3.4 ppm. For methylcarbonates and acetates, signals, overlapping with methylene groups neighboring nitrogen atoms within linkers, were visible at 3.45-3.65 ppm. Values of critical micelle concentrations for three groups of surfactants, i.e. methylcarbonates, bromides and acetates, followed the Stauff-Klevens rule and were within the range 10 -2 -10 -4 M for C12-C16 derivatives, corresponding to typical values of linear-type ionic surfactants 28 .

Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC).
Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined to evaluate the activity of the newly synthesized QAS. The tested compounds differed in the structure of the counterion (Me-methylcarbonate, Ac-acetate, Br-bromide) and the length of the aliphatic chain (C12, C14, C16). The activity of the compounds was analyzed against Gram-positive strains: S. epidermidis ATCC 35984, S. epidermidis B374, S. epidermidis SI8, S. aureus ATCC 6538, S. aureus MRSA R98 and Gram-negative: P. aeruginosa ATCC 27853, P. aeruginosa PAO1, E. coli ATCC 11229 and E. coli H64.
The structure of the surfactant was shown to affect its antimicrobial activity-the compounds with 14 and 16 carbon atoms in the aliphatic chain were more effective against Gram-positive bacteria that those with 12 carbon atoms. Among Gram-negative bacteria P. aeruginosa ATCC 27,853 and clinical strain were less susceptible to tested chemicals. As regards type of counterion, the most effective were methylcarbonate and bromide with C16 hydrophobic chains. Tested compounds were less effective against Gram-negative bacteria. The lowest MIC was obtained for E. coli H64, which for the MeC14 and MeC16 compounds was 80 µM. Against E. coli ATCC 11,229, acetate with 16 carbon atoms in the chain showed the greatest effectiveness. It caused growth inhibition of the strain at 80 µM. None of the QAS tested showed effectiveness for P. aeruginosa strains from the ATCC collection and the clinical strain. Among Gram-positive bacteria the most sensitive were S. epidermidis B374, S. epidermidis SI8, S. aureus ATCC 6538, S. aureus MRSA R98 against which the compounds with 16 carbon atoms in the chain showed activity in concentrations of 10 µM (p < 0.004) ( Table 1).   3,4,5,6,7,8,9,10). There was no correlation between the structure of the counterion and the anti-adhesion effect. It was observed that methylcarbonates with a 16-carbon aliphatic chain (MeC16) inhibited the adhesion to the stainless steel surface of S. epidermidis ATCC 35984, S. epidermidis B374, and P. aeruginosa PAO1 at a level of almost 69-83% (p < 0.004). Reduced adhesion to the polystyrene surface after treatment with MeC16 at a concentration of 320 µM (0.03 CMC) was exhibited by S. epidermidis B374. The reduction in adhesion was estimated at the level of 77% (p < 0.004) (Fig. 2).
Furthermore, it was found that the application of the compound (BrC16) inhibited the adhesion of S. epidermidis B374 to the polystyrene surface by approximately 85% at a concentration of 160 µM (0.2 CMC) (p = 0.04). This compound did not reduce the adhesion of the other strains to this surface (Fig. 8).
Adhesion to silicone and glass is described in Electronic Supplementary Material (Figs. S1-S9). Adhesion to these surfaces, in most cases, stayed high, regardless of the used QAS. Tested compounds inhibited adhesion to silicone at the level of 20-40% (p < 0.004). MeC16, MeC12, BrC16, BrC14, and BrC12 reduced adhesion P. aeruginosa PAO1 to glass by approximately 20% (p < 0.004).
Biofilm eradication. QAS are capable of destroying mature bacterial and fungal biofilms. These compounds penetrate the formed biofilm and destroy biofilm structure.
No correlation was observed between the structure of the counterion and the biofilm eradication activity. However, compounds with a methylcarbonate counterion showed less activity (Fig. 11). The study showed that

Biofilm-oriented antiseptics test (BOAT).
Among disinfectants, compounds are not only effective against microorganisms in low concentrations but are also characterized by fast action. For this purpose, the biofilm-oriented antiseptics test (BOAT) was performed. It was found that the analyzed compound (AcC16) showed efficacy against S. epidermidis B374 biofilm after 30 min of incubation (640 µM; 0.2 CMC), causing a decrease in cell survival of up to 30% and up to 9.5% after 60 min (Fig. 13). AcC16 was chosen for its most effective activity against S. epidermidis B374, P. aeruginosa PAO1.

Mutagenicity.
To evaluate the potential future application of the compounds, an analysis of their mutagenic properties is necessary. It was observed that among the nine QAS tested with different counterions (Me, Ac, Br) and chain lengths (C12, C14, C16), only BrC12 and BrC16 caused a reading frame shift type mutation. Their mutagenicity index against the strain S. Typhimurium TA98 was above 1.7 (MR > 1.7). This indicates the potential mutagenic properties of the QAS analyzed. These compounds did not cause base pair substitution type mutations, as their mutagenicity index against S. Typhimurium TA100 did not exceed the value of 1.7 (MR < 1.7). Other compounds tested do not have mutagenic potential (Table 2).
Hemolytic properties. Another necessary study for the applied use of the compounds is the analysis of the hemolytic properties of the tested QAS. Determination of hemolytic properties against animal erythrocytes is one of the most common tests in preliminary studies of chemicals with blood components 29 . Compounds with counterions such as methylcarbonate (Me), acetate (Ac) and bromide (Br) and chain lengths of C12, C14, C16 were tested. The analyses showed that the tested surfactants induced hemolysis of sheep cells only at high concentrations (80-1280 µM). The concentrations at which hemolysis was observed often exceeded the MIC concentrations of the compounds. It was observed that the longer the aliphatic chain of the compound was, the greater was the hemolysis of blood cells, regardless of the type of the counterion (Fig. 14).

Discussion
Monomeric QAS are a class of surfactants composed of a single head and a single aliphatic chain. In their structure they contain a counterion which often increases the effect of the compound 8 . They show biological activity against pathogenic microorganisms not only in planktonic forms, but also against biofilms 30 . Due to the excessive use of surfactants, there is a problem of increasing resistance to disinfectants among microorganisms, including quaternary ammonium salts 31 . The current work is an approach to select the most effective QAS in terms of antimicrobial activity against planktonic forms and biofilms. In addition, compounds inhibiting adhesion to the surface of stainless steel, polystyrene, glass, and silicone are also sought. The surfactants used in this work differed in aliphatic chain length (C12, C14, C16) and type of counterion (methylcarbonate, acetate, bromide). This selection allowed us to separate compounds which will be subjected to further stages of research, i.e., molecular analysis of mechanisms of action against Saccharomyces cerevisiae cells. In general, studies carried out on surfactants suggest that their main mechanisms involve perforation of cell membranes and cell disruption. However, it is reported 9,32 that some QAS do not cause perforation of cell membranes but interact with them by changing their permeability. These interactions can lead to disruption of plasma membrane structure, resulting in leakage of intracellular material or alteration of plasma membrane function (e.g., proton gradient or secondary transport). Moreover, they can cause oxidative stress or affect lipid metabolism, resulting in accumulation of lipid droplets in the cytosol. Thus, One of the structural elements that has a great influence on the activity of the compounds is the length of the aliphatic chain. Mono-QAS with short chains (C2, C3) do not show biological activity. On the other hand, the optimal length of the hydrophobic carbon chain of compounds is determined from 12 to 18 carbon atoms 10 . The long alkyl chain can be built into the plasma membrane of microorganisms, while short chain surfactants may not have this ability. The highest effectiveness of QAS with long alkyl chain was observed against Gram-positive bacteria. Among Gram-negative bacteria, low activity of the compounds was observed, which is consistent with literature reports where bacterial envelopes are more difficult for the compounds to penetrate 10 . Reduced susceptibility among Gram-negative bacteria is determined not only by the presence of an outer membrane and lipopolysaccharide, but also by multiple resistance mechanisms, such as efflux pumps 33 . On the other hand, some studies suggest the compounds have activity against Gram-negative bacteria, fungi, and parasites 34,35 . Our study indicates that the antimicrobial activity of mono-QAS increases with aliphatic chain length. This observation is consistent with previous literature 35,36 . As reported by Gozzelino et al. 30 , mono-QAS with 16 carbon atoms in the chain showed greater activity against planktonic forms of Escherichia coli, Staphylococcus aureus and Listeria monocytogenes relative to compounds with shorter chains. Interestingly, this phenomenon is seen not only among mono-QAS but also among multifunctional compounds. Paluch et al. 32 reported that multifunctional QAS with longer alkyl chains (C14, C16) exhibited greater antimicrobial potential. It is intriguing to note that among other gemini compounds, often higher activity was observed with shorter chain length 11,22 . These QAS have an additional head, an aliphatic chain, and a linker, which affect their ability to penetrate bacterial cell envelopes, which impact the antimicrobial activity of surfactants.
The strong cohesion between hydrophobic chains may be connected with presence of the secondary amide linker moiety with three methylene groups connecting the headgroup with the spacer, virtually lengthening the surfactant tail group. Presence of an N-H group in the secondary amide linker may also lead to occurrence of hydrogen bonding, resulting in strong cohesive forces between chains. Generally, moderation of strong hydrogen bonding, resulting in inferior water solubility and high values of Krafft points, i.e. the minimal temperature at which the surfactant exhibits sufficient activity in aqueous systems, may be performed by replacement of the secondary (i.e. containing an N-H bond) by a tertiary amide (e.g. containing an N-Me motif) moiety. The mentioned effect was widely studied for amphoteric 2-hydroxypropanesulfobetaine-type alkylamidopropyl derivatives 28 .
The most well-known, and probably the most commercially viable, example of cleavable surfactants comprises the family of cationic esterquat-type surfactants (often abbreviated as esterquats) with the ester bond -CO-O-or -O-CO-located between the quaternary ammonium head group(s) and the hydrocarbon tail(s) [37][38][39] . Grafting of the hydrolysable ester moiety onto the hydrophobic backbone of the surfactant structure allows decomposition of the molecule into fragments, lowering the environmental exposure levels and, furthermore, making it possible to improve the rate of biodegradation and to obtain high quality, environmentally friendly products for various www.nature.com/scientificreports/ applications. Moreover, using hydrolysable surfactants for formation of drug nanocarriers opens the possibility of designing new controlled delivery systems that can be activated by an internal or external triggering mechanism, as they are stable in aqueous solutions for a certain period only within a certain pH range. Amide bonds, like ester bonds, can be cleaved either by chemical hydrolysis or by enzyme catalyzed hydrolysis 15,37 . The amide bond is more stable than the ester bond to alkaline hydrolysis but is usually more susceptible to acid catalyzed cleavage. Amidases and peptidases are examples of amide-splitting enzymes. Lipases, which are normally associated with ester bond cleavage, sometime work also on amide bonds 40 . The chemical stability of the amide bond was found to be high. When the surfactant was subjected to 1 M sodium hydroxide for five days at room temperature, only 5% was cleaved. The corresponding experiment performed in 1 M HCl resulted in no hydrolysis. The amide bond was, however, found to be slowly hydrolyzed when lipase from Candida antarctica or peptidase was used as a catalyst. Amidase and lipase from Mucor miehei were found to be ineffective. Despite the very high chemical stability, the amide surfactant biodegraded by a similar path in the plot of biodegradation versus time as the corresponding ester surfactant, reaching 60% biodegradation within 28 days. Hence, it can be classified as readily biodegradable 40 . The mentioned facts clearly show that an amide linker in surfactants constitutes a good compromise between usefulness (i.e. chemical stability in basic, neutral and acidic conditions during storage and usage) and appropriate susceptibility to undergo biodegradation after use or in case of release to the environment. Monomeric QAS are generally considered to be less active compared to multifunctional or gemini forms 10,11,41 . In the current study, compounds with 16 carbon atoms show efficacy against Gram-positive bacteria at low concentrations (MIC = 10 µM). The efficacy of 12-and 14-carbon compounds was lower. Compounds with longer aliphatic chains (C14, C16) also eradicated biofilms more effectively. Such a trend was also seen among multifunctional compounds, where compounds with 16 carbon atoms in the aliphatic chain better eradicated C. albicans biofilm 32 . This might be due to the fact that they penetrated and destabilized the biofilm matrix more effectively than compounds with shorter chains (C12).
QAS in low concentrations, below the CMC value, can form tiny aggregates called premicelles, which may decrease the activity of the compounds by reduction of their mobility.
Moreover, culture medium containing amphoteric components may influence this process. The activity of quaternary ammonium salts is enhanced by counterions, in particular reactive ions, i.e. those having the ability to oxidize proteins or lipids. Among the most active are compounds possessing chlorine, bromide, or iron atoms 33,36,42 . In contrast, studies indicate that the counterions of mono-QAS compounds are not www.nature.com/scientificreports/ as important as the aliphatic chain length for the activity of the compound 36 . In our analyses, there was no clear effect of the nature of the counterion on antimicrobial activity against planktonic forms. However, mono-QAS having acetate or bromide in their structure were slightly more effective in eradicating the biofilm of all tested strains. In addition, bromide in the counterion most likely enhanced the mutagenic properties of compounds with 12 and 16 carbon atoms in the chain. Preventing the adhesion of microorganisms to surfaces is one method to prevent biofilm formation on abiotic surfaces. QAS have the ability to coat surfaces such as stainless steel, polystyrene, glass or silicone. These materials are used for catheters, endoprostheses, stents, and pacemakers, among other applications 19 . Therefore, it is extremely important to maintain their sterility throughout their lifetime 43,44 . One potential candidate is QAS or polymeric coatings containing immobilized QAS molecules 21 . Such a solution was applied by Kok et al. 45 Application of quaternary ammonium silane significantly reduced Enterococcus faecalis and Candida albicans biofilm 45 . Studies have shown that compounds with longer chains (> C8) were generally more effective in preventing microbial adhesion. In the present study, methylcarbonates, acetates, and bromides with 16-carbon aliphatic chains (MeC16, AcC16 and BrC16) showed the highest efficacy among the nine compounds tested. This is most likely because most surfaces are naturally hydrophobic, so they interact with the hydrophobic aliphatic chain. The hydrophobicity of the compound will increase with the length of its chain 36,46 . As Paluch et al. point out 32 the hydrophilic head of the compound via electrostatic interactions can repel the hydrophobic surface of the microorganism cell. Another possibility is the interaction of the cationic head of the compound with a negatively charged plasma membrane, leading to a change in the cell surface potential, thereby causing a disruption in its functioning 32 . Also, the research by Gozzelino et al. 30 showed that mono-QAS with 16 carbon atoms in the chain effectively inhibited the adhesion of microorganisms to stainless steel. A compound with 8 carbon atoms in the chain also showed high effectiveness in their study 30 . Interactions that occur at the surface-QAS interface are the focus of surfactant research, as understanding the mechanisms of action is key to their application. It should be mentioned that some QAS can stimulate adhesion of microorganisms to surfaces, as has been reported by Paluch et al., Machado et al.,and Ortiz et al. 32,47,48 .
For the future application of the compounds as medical plastic coatings, several studies are required that, among other things, would rule out their toxicity to higher organisms. Such studies include mutagenicity analysis and assessment of hemolytic properties of the compounds. Mono-QAS are considered to be non-toxic, biodegradable and environmentally safe compounds. Hence, in the form of ionic liquids they are called "green solvents". They are less toxic than dimeric, trimeric, or polymeric forms 10 . However, due to their nature, surfactants can penetrate the cell, thereby changing the osmolarity and causing cell lysis. Numerous studies indicate that the length of the aliphatic chain affects not only the antimicrobial activity of compounds, but also their toxicity 10,[49][50][51] . In our study, hemolysis performed on animal blood cells is seen only at high compound concentrations exceeding the MIC. A similar result regarding other QAS was obtained by Xie et al. 52 , who noted a relationship between hemolytic potential and surfactant concentration 52 . According to the literature, it has also been observed that the longer the aliphatic chain is, the greater are the hemolytic properties. Therefore, it is necessary to invent compounds with optimal chain length in terms of toxicity to cells of higher organisms as well as antimicrobial activity. For example, Florio et al. 51,53 observed that 12-carbon compounds with bromide in their structure (N-dodecyl-N-methylpyrrolidinium bromide, N-dodecyl-N-methylpiperidinium bromide) are characterized by high activity and low toxicity, which allowed application of the compounds in medicine [36] [38]. Novel custom designed QAS type surfactants were designed and synthesized, providing such optimal structural parameters as the following: labile tertiary amide linker, able to moderate the hydrogen bonding within the surfactant molecule and to increase the aqueous solubility, varying hydrophobic tail and different counterions, including soft and reactive ones, good chemical stability, and potential biodegradability. The preparation methodology fulfills at least some principles of "green chemistry"-for application as disinfectants.

Materials and methods
Materials. Chemicals. All the used reagents (reagent or analytical grade) were used as received and obtained from Sigma-Aldrich, except N,N,N′-trimethyl-1,3-propanediamine (Alfa Chemistry). Deuterated solvents were purchased from Sigma-Aldrich. Organic solvents (analytical grade) were obtained from Avantor Performance Materials. Triethylamine was dried with solid calcium hydride (1 g per 50 mL of solvent) followed by distillation over this drying agent. Water used in all experiments was double-distilled (synthetic procedures) or purified by a Millipore (Bedford, MA) miliQ purification system (conductometric measurements). Methods. 1 H NMR analysis. 1 H NMR spectra were recorded on a Bruker AMX-500 spectrometer, using CDCl3 (chloroform-d) as a solvent (Tables 3, 4, 5). 1 H chemical shifts (in ppm) were calibrated to TMS as an internal reference.

Minimal Inhibitory Concentration (MIC) and Minimal Bactericidal Concentration (MBC).
The minimum inhibitory concentration (MIC) of the tested monomeric QAS was performed using the microdilution method according to CLSI recommendation (M27-A3). Concentrations from 10 to 1280 µM were analyzed. Strains were incubated with or without compounds (growth control) for 24 h at 37 °C. Three independent replicates were performed. The MIC value was determined spectrophotometrically and the concentration that inhibited more than 90% of growth was determined. Optical density was measured at λ = 590 nm using a 96-well microplate reader (Bio-Rad Universal Hood II).
To determine the minimum bactericidal concentration (MBC), 10 µL of bacterial suspension incubated with monomeric QAS (MIC concentration and two times higher) was transferred to plates with LB (1% tryptone, 1% yeast extract, 0.5% NaCl) medium. Plates were incubated at 37 °C for 24 h and then colonies were counted. The concentration that reduced 99.9% of microbial growth was determined as MBC 54 .
Adhesion. Adhesion to polystyrene plate. Adhesion analysis to polystyrene surfaces was determined according to the method of Rodrigues et al. with modifications 55 . Polystyrene 96-well plates were incubated with the respective compound concentrations (10-1280 µM) for 4 h at 37 °C with 100 rpm shaking. The plates were then washed with miliQ and inoculated with 100 µL of bacterial suspension (OD = 0.4-0.6). Plates were incubated for 6 h at 37 °C with 100 rpm shaking. Wells were rinsed and fixed for 20 min at 60 °C. They were then decolorized with 0.1% crystal violet solution for 5 min. The wells were washed with miliQ until the unfixed dye was completely eluted. The absorbance at λ = 590 nm was measured using a reader (Bio-Rad Universal Hood II). The positive control was a surface not treated with QAS. Three independent trials were performed.  57 . A standardized bacterial (OD = 0.6) suspension of 100 µL was spotted into a 96-well polystyrene plate. They were incubated for 24 h at 37 °C. Cultures were removed, the plate was washed and then concentrations of QAS (10-1280 µL) were added, incubated for 4 h at 37 °C with 100 rpm shaking. The wells were washed with miliQ and fixed at 60 °C for 20 min. The wells were decolorized with 0.1% crystal violet solution for 5 min. The wells were washed with miliQ until the unfixed dye was completely eluted. The absorbance at λ = 590 nm was measured using a reader (Bio-Rad Universal Hood II). The positive control was an untreated biofilm. Three independent test trials were performed.
Biofilm-oriented antiseptics test (BOAT). Biofilm-oriented antiseptics test (BOAT) was performed according to the method of Junka et al. 58 . The strain (S. epidermidis B374) was cultured into an appropriate liquid TSB medium and incubated at 37 °C for 24 h. After incubation, the bacterial suspension was diluted with fresh medium to optical density OD = 0.125 (λ = 590). A dilution 10 -3 was made a and transferred to 96-well polystyrene plate. Next, the suspensions were incubated at 37 °C for 24 h. After 24 h, the suspensions from both plates were removed and thoroughly rinsed with 0.9% NaCl. Next, 100 μL of undiluted (working solution) of QAS (AcC16) in a certain concentration was transferred to the well for selected contact time (30 min and 60 min). After the contact time, the antiseptic was removed and the wells were filled with an appropriate universal neutralizing agent (Saline Peptone Water, Biocorp, Warsaw, Poland) for 5 min. After this time, the neutralizing agent was removed. The wells were filled with 100 μL of an appropriate medium and with 5 μL of tetrazolium chloride (TTC) (Fluka, Poznan, Poland), a reagent staining metabolically active microorganisms red. The results were assessed colorimetrically after 24 h of incubation of the plate at 37 °C. Samples were diluted and suspended 100 µL per LB and incubated at 37 °C. Colonies were then counted. The positive control was an untreated biofilm, confirming the ability of the test strain to form biofilm. The negative control wells filled with medium (no biofilm) confirming the sterility of the test performer and TTC.
Mutagenicity. Two reference strains of Salmonella Typhimurium TA98 and Salmonella Typhimurium TA100, deficient in histidine synthesis, were used for this study according to the method proposed by Ames et al. with modifications 59 . 100 µL bacterial culture (OD = 1.5), 100 µL compound of a specific concentration (1/2 MIC, 1/4 MIC) and 200 µL biotin (0.031%) with histidine (0.024%) solution were added to 2 mL top agar. The mixture was mixed and poured onto a Davis minimal medium plate. The mixture without the test compound was used as a negative control. The positive control was a mixture of bacterial culture, biotin-histidine solution, and sodium azide (at a concentration of 15 μg/mL for strain TA100) or acriflavine (at a concentration of 100 μg/mL for strain TA98). Plates were incubated for 48 h at 37 °C, and then colonies were counted. The mutagenicity ratio (MR), the ratio of the number of revertants grown in the presence of the test compound to the number of spontaneously occurring revertants (negative control), was calculated. A mutagenic ratio equal to or greater than 1.7 indicates the mutagenic potential of the tested compounds. The study was performed in three times.
Hemolytic properties. QAS was assayed for hemolytic activity as described by Falkinham III et al. 60 . Sheep blood (5 mL) was centrifuged for morphotic elements (2500 rpm, 15 min), washed three times in PBS (pH 7.4) and resuspended in PBS. Compounds of different concentrations in a volume of 10 µL (2.5 -1280 µL) were transferred to the wells of a titer plate and mixed with 90 µL of erythrocytes. They were incubated for 1.5 h at 37 °C. Centrifuged (2500 rpm, 15 min), and the supernatant was transferred to a sterile flat-bottomed titer plate. The absorbance at λ = 540 was measured (Bio-Rad Universal Hood II). PBS and 1% SDS were used as positive and negative controls, respectively. This assay was repeated three times.

Statistical analysis.
The results of all the experiments are given as a mean value ± SD (standard deviation) of three independent experiments. The differences in adhesion to different surfaces, biofilm eradication, hemolysis properties of QAS were analyzed with a parametric t-test for independent samples using Statistica v. 13 software (StatSoft, Krakow, Poland). Differences between groups were considered statistically significant for p values < 0.05.

Data availability
The datasets used and/or analysed during the current study available from the corresponding author on reasonable request. All samples are available from the authors upon request.